cDNA cloning and functional analysis of ascidian sperm proacrosin

cDNA cloning and functional analysis of proacrosin from the ascidian Halocy nthia roretzi were undertaken. The isolated cDNA of the ascidian preproacro sin consists of 2367 nucleotides, and an open reading frame encodes 505 ami no acids, which corresponds to the molecular mass of 55,003 De. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization, The amino acid sequences around His(76), Asp(132), and Ser(227), which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys(56)-His(57)) in the N-term inal region, which is one of the most characteristic features of mammalian acrosin, This region seems to play a key role in the binding of (pro)acrosi n to the vitelline coat, because the peptide containing the paired basic re sidues, but not the peptide substituted with Ale, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin conta ins two CUB domains in the C-terminal region, in which CUB domain 1 seems t o be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin we re identified. In response to sperm activation, acrosin was released from s perm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate th at ascidian sperm contains a mammalian acrosin homologue, a multi-functiona l protein working in fertilization.